Abstract

The performance of third-harmonic generation (THG) microscopy in highly scattering media is analyzed with the Monte Carlo technique. The three-dimensional point-spread function (PSF) of the laser-scanning THG microscope with a pulsed excitation light source is derived for both isotropic and anisotropic scattering media and at different h/ d s values, where h is the scattering depth as measured from the geometric focus of the objective lens and d s is the mean free path of the scattering medium. The generated THG signal is detected by a large-area photodetector. The PSF of the THG microscope is given by the third power of the normalized distribution of the excitation beam near the beam focus. The behavior of the temporal broadening of the excitation pulse on the generated THG signal is also analyzed as a function of h/ d s. The relative advantages and disadvantages of the THG microscope relative to the two-photon fluorescence microscope are discussed thoroughly.

© 2000 Optical Society of America

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References

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  1. J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
    [CrossRef]
  2. D. Yelin, Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Exp. 5, 169–175 (1999).
    [CrossRef]
  3. Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
    [CrossRef]
  4. D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
    [CrossRef]
  5. M. Muller, J. Squier, G. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
    [CrossRef] [PubMed]
  6. J. A. Squier, M. Müller, “Third-harmonic generation imaging of laser induced breakdown in glass,” Appl. Opt. 38, 5789–5794 (1999).
    [CrossRef]
  7. T. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
    [CrossRef] [PubMed]
  8. C. Palmes-Saloma, C. Saloma, “Long depth imaging of specific gene expressions in wholemount mouse embryos with single-photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology (to be published).
  9. C. Blanca, C. Saloma, “Monte-Carlo analysis of two-photon imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998).
    [CrossRef]
  10. C. Blanca, C. Saloma, “Efficient analysis of temporal broadening of a pulsed focused Gaussian beam in scattering media,” Appl. Opt. 38, 5433–5437 (1999).
    [CrossRef]
  11. R. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).
  12. A. Siegman, Lasers (University Science Books, Mill Valley, Calif., 1986).
  13. V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
    [CrossRef]
  14. E. Wolf, M. Born, Principles of Optics, 6th ed. (Pergamon, New York, 1980), pp. 647–656.
  15. V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.
  16. W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
    [CrossRef]
  17. C. Saloma, M. O. Cambaliza, “Single Gaussian beam interaction with a dielectric microsphere: radiation force, multiple internal reflections and caustic structures,” Appl. Opt. 34, 3522–3528 (1995).
    [CrossRef] [PubMed]
  18. C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Bio. 47, 1741–1759 (1998).
    [CrossRef]

1999 (4)

D. Yelin, Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Exp. 5, 169–175 (1999).
[CrossRef]

D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
[CrossRef]

J. A. Squier, M. Müller, “Third-harmonic generation imaging of laser induced breakdown in glass,” Appl. Opt. 38, 5789–5794 (1999).
[CrossRef]

C. Blanca, C. Saloma, “Efficient analysis of temporal broadening of a pulsed focused Gaussian beam in scattering media,” Appl. Opt. 38, 5433–5437 (1999).
[CrossRef]

1998 (5)

V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
[CrossRef]

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Bio. 47, 1741–1759 (1998).
[CrossRef]

J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
[CrossRef]

C. Blanca, C. Saloma, “Monte-Carlo analysis of two-photon imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998).
[CrossRef]

M. Muller, J. Squier, G. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[CrossRef] [PubMed]

1997 (1)

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

1995 (2)

1990 (1)

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Barad, Y.

D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
[CrossRef]

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Blanca, C.

Born, M.

E. Wolf, M. Born, Principles of Optics, 6th ed. (Pergamon, New York, 1980), pp. 647–656.

Brakenhoff, G.

M. Muller, J. Squier, G. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[CrossRef] [PubMed]

Brakenhoff, G. J.

J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
[CrossRef]

Cambaliza, M. O.

Cheong, W.

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Daria, V.

V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
[CrossRef]

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Fujita, K.

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Kawata, S.

V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
[CrossRef]

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

Kondoh, H.

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Bio. 47, 1741–1759 (1998).
[CrossRef]

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

Muller, M.

M. Muller, J. Squier, G. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[CrossRef] [PubMed]

Müller, M.

J. A. Squier, M. Müller, “Third-harmonic generation imaging of laser induced breakdown in glass,” Appl. Opt. 38, 5789–5794 (1999).
[CrossRef]

J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
[CrossRef]

Nakamura, O.

V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
[CrossRef]

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

Newton, R.

R. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).

Palmes-Saloma, C.

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Bio. 47, 1741–1759 (1998).
[CrossRef]

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

C. Palmes-Saloma, C. Saloma, “Long depth imaging of specific gene expressions in wholemount mouse embryos with single-photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology (to be published).

Patel, J.

D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
[CrossRef]

Prahl, S.

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Saloma, C.

C. Blanca, C. Saloma, “Efficient analysis of temporal broadening of a pulsed focused Gaussian beam in scattering media,” Appl. Opt. 38, 5433–5437 (1999).
[CrossRef]

C. Blanca, C. Saloma, “Monte-Carlo analysis of two-photon imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998).
[CrossRef]

V. Daria, C. Blanca, O. Nakamura, S. Kawata, C. Saloma, “Image contrast enhancement for two-photon fluorescence microscopy in turbid medium,” Appl. Opt. 37, 7960–7967 (1998).
[CrossRef]

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Bio. 47, 1741–1759 (1998).
[CrossRef]

C. Saloma, M. O. Cambaliza, “Single Gaussian beam interaction with a dielectric microsphere: radiation force, multiple internal reflections and caustic structures,” Appl. Opt. 34, 3522–3528 (1995).
[CrossRef] [PubMed]

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

C. Palmes-Saloma, C. Saloma, “Long depth imaging of specific gene expressions in wholemount mouse embryos with single-photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology (to be published).

Siegman, A.

A. Siegman, Lasers (University Science Books, Mill Valley, Calif., 1986).

Silberberg, Y.

D. Yelin, Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Exp. 5, 169–175 (1999).
[CrossRef]

D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
[CrossRef]

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Squier, J.

M. Muller, J. Squier, G. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[CrossRef] [PubMed]

Squier, J. A.

J. A. Squier, M. Müller, “Third-harmonic generation imaging of laser induced breakdown in glass,” Appl. Opt. 38, 5789–5794 (1999).
[CrossRef]

J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
[CrossRef]

Tsang, T.

T. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
[CrossRef] [PubMed]

Welch, A.

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Wilson, K. R.

J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
[CrossRef]

Wolf, E.

E. Wolf, M. Born, Principles of Optics, 6th ed. (Pergamon, New York, 1980), pp. 647–656.

Yelin, D.

D. Yelin, Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Exp. 5, 169–175 (1999).
[CrossRef]

D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
[CrossRef]

Appl. Opt. (5)

Appl. Phys. Lett. (1)

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

IEEE J. Quantum Electron. (1)

W. Cheong, S. Prahl, A. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

J. Microsc. (1)

M. Muller, J. Squier, G. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[CrossRef] [PubMed]

Opt. Exp. (2)

J. A. Squier, M. Müller, G. J. Brakenhoff, K. R. Wilson, “Third harmonic generation microscopy,” Opt. Exp. 3, 315–324 (1998).
[CrossRef]

D. Yelin, Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Exp. 5, 169–175 (1999).
[CrossRef]

Phys. Med. Bio. (1)

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Bio. 47, 1741–1759 (1998).
[CrossRef]

Phys. Rev. A (1)

T. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
[CrossRef] [PubMed]

Phys. Rev. Lett. (1)

D. Yelin, Y. Silberberg, Y. Barad, J. Patel, “Phase-matched third-harmonic generation in a nematic liquid crystal cell,” Phys. Rev. Lett. 82, 3046–3049 (1999).
[CrossRef]

Other (5)

R. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).

A. Siegman, Lasers (University Science Books, Mill Valley, Calif., 1986).

C. Palmes-Saloma, C. Saloma, “Long depth imaging of specific gene expressions in wholemount mouse embryos with single-photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology (to be published).

E. Wolf, M. Born, Principles of Optics, 6th ed. (Pergamon, New York, 1980), pp. 647–656.

V. Daria, C. Palmes-Saloma, K. Fujita, C. Saloma, O. Nakamura, H. Kondoh, S. Kawata, “Long depth imaging of turbid biological samples by two-photon microscopy,” in Proceedings of the Nineteenth Meeting of the Japan Society for Laser Microscopy (Japan Society for Laser Microscopy, Tokyo, 1997), pp. 28–32.

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Figures (9)

Fig. 1
Fig. 1

Optical setup of the laser-scanning THG microscope. The Dirac-delta excitation pulse of wavelength λ e is produced by the point light source S and focused toward the point S′ at z = 0 by L2. An intervening nonabsorbing medium exists between L1 and the sample plane. The THG signal is emitted from S′ and collected by the large-area photodetector. The optical filter F permits only the THG photons of wavelength λ g = λ e /3 to reach the photodetector. Points S, S′, and S″ are conjugate points. Full description of the propagation process is described in the text.

Fig. 2
Fig. 2

Log-scale plot of stability factor ρ with h/d s for N e = 103 (filled square), 104 (filled circle), 105 (circle), 106 (cross hair), and 107 (square). Parameters used are NA = 0.6, and g = 0.001. For the same h/d s value, an order of improvement in ρ is only achieved when N e is increased by an order of magnitude.

Fig. 3
Fig. 3

Isotropic scattering (g = 0.001). Time-integrated distribution of l e (r, z) near the focus of L2 for the following h/d s values: (a) 2, (b) 4, (c) 6, (d) 8. The beam is propagated from the top to the bottom (NA = 0.6), and the dashed lines represent the boundary of the geometric shadow. Scattering broadens the spatial distribution of l e (r, z) with its peak shifting toward the side of L2. The l e (0, z) distribution broadens asymmetrically about the focus of L2. On the other hand, the l e (r, 0) distribution is robust against the ill effects of isotropic scattering. Frame size, 500 µm × 500 µm.

Fig. 4
Fig. 4

Anisotropic scattering (g = 0.9). Time-integrated distribution of l e (r, z) near the focus of L2 for the following h/d s values: (a) 2, (b) 4, (c) 6, (d) 8. Scattering broadens the spatial distribution of l e (r, z) with its peak shifting toward the side of L2. Both the l e (0, z) and the l e (r, 0) distributions broaden rapidly with increasing h/d s values. Frame size, 500 µm × 500 µm.

Fig. 5
Fig. 5

Plots of l e (0, z) at (a) g = 0.001 and (b) g = 0.9 for h/d s = 2 (solid curve), 4 (dotted curve), 6 (filled circle), and 8 (cross hair), where NA of L2 = 0.6 (focal length of L2 = 4.3 mm).

Fig. 6
Fig. 6

Plots of the generated THG signal l g (0, z) = l e 3(0, z) at (a) g = 0.001 and (b) g = 0.9, for h/d s = 2 (solid curve), 6 (filled circle), and 8 (cross hair), where NA of L2 = 0.6 (focal length of L2 = 4.3 mm).

Fig. 7
Fig. 7

Intensity plots of l e 2(0, z) at (a) g = 0.001 and (b) g = 0.9, for h/d s = 2 (solid curve), 6 (filled circle), and 8 (cross hair), where NA of L2 = 0.6 (focal length of L2 = 4.3 mm).

Fig. 8
Fig. 8

Comparison of the 2P intensity distribution l e 2(0, z) (thin curves) and the THG signal (thick curves) l e 3(0, z) for h/d s = 8. (a) For g = 0.9, the THG process suppressed the background to half of its 2P level. (b) Relative advantage of both processes for an isotropic medium (g = 0.001) is apparent.

Fig. 9
Fig. 9

Peak value l p of the excitation pulse at z = 0, as a function of h/d s . For g = 0.001 (filled circles), the decrease in the peak excitation intensity is exponential and can be described by (best-fit curve) l p = 0.929exp(-0.953h/d s ). For g = 0.9, the decrease in l p may be described by a power law (best-fit curve) l p = 0.197(h/d s )-1.784.

Equations (2)

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cos θj=2g-11+g2-1-g222g-11-g+2gσ2,
lgx, y, z=le3x, y, z,

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